BackgroundThe detection and characterization of resistance reactions of crop plants against fungal pathogens are essential to select resistant genotypes. In breeding practice phenotyping of plant genotypes is realized by time consuming and expensive visual rating. In this context hyperspectral imaging (HSI) is a promising non-invasive sensor technique in order to accelerate and to automate classical phenotyping methods.A hyperspectral microscope was established to determine spectral changes on the leaf and cellular level of barley (Hordeum vulgare) during resistance reactions against powdery mildew (Blumeria graminis f.sp. hordei, isolate K1). Experiments were conducted with near isogenic barley lines of cv. Ingrid, including the susceptible wild type (WT), mildew locus a 12 (Mla12 based resistance) and the resistant mildew locus o 3 (mlo3 based resistance), respectively. The reflection of inoculated and non-inoculated leaves was recorded daily with a hyperspectral linescanner in the visual (400 – 700 nm) and near infrared (700 – 1000 nm) range 3 to 14 days after inoculation.ResultsData analysis showed no significant differences in spectral signatures between non-inoculated genotypes. Barley leaves of the near-isogenic genotypes, inoculated with B. graminis f.sp. hordei differed in the spectral reflectance over time, respectively. The susceptible genotypes (WT, Mla12) showed an increase in reflectance in the visible range according to symptom development. However, the spectral signature of the resistant mlo-genotype did not show significant changes over the experimental period. In addition, a recent data driven approach for automated discovery of disease specific signatures, which is based on a new representation of the data using Simplex Volume Maximization (SiVM) was applied. The automated approach - evaluated in only a fraction of time revealed results similar to the time and labor intensive manually assessed hyperspectral signatures. The new representation determined by SiVM was also used to generate intuitive and easy to interpretable summaries, e.g. fingerprints or traces of hyperspectral dynamics of the different genotypes.ConclusionWith this HSI based and data driven phenotyping approach an evaluation of host-pathogen interactions over time and a discrimination of barley genotypes differing in susceptibility to powdery mildew is possible.

Cercospora leaf spot (CLS) caused by Cercospora beticola is the most destructive leaf disease of sugar beet and may cause high losses in yield and quality. Breeding and cultivation of disease-resistant varieties is an important strategy to control this economically relevant plant disease. Reliable and robust resistance parameters are required to promote breeding progress. CLS lesions on five different sugar beet genotypes incubated under controlled conditions were analyzed for phenotypic differences related to field resistance to C. beticola. Lesions of CLS were rated by classical quantitative and qualitative methods in combination with noninvasive hyperspectral imaging. Calculating the ratio of lesion center to lesion margin, four CLS phenotypes were identified that vary in size and spatial composition. Lesions could be differentiated into subareas based on their spectral characteristics in the range of 400 to 900 nm. Sugar beet genotypes with lower disease severity typically had lesions with smaller centers compared with highly susceptible genotypes. Accordingly, the number of conidia per diseased leaf area on resistant plants was lower. The assessment of lesion phenotypes by hyperspectral imaging with regard to sporulation may be an appropriate method to identify subtle differences in disease resistance. The spectral and spatial analysis of the lesions has the potential to improve the screening process in breeding for CLS resistance.

The quantitative resistance of sugar beet (Beta vulgaris L.) against Cercospora leaf spot (CLS) caused by Cercospora beticola (Sacc.) was characterised by hyperspectral imaging. Two closely related inbred lines, differing in two quantitative trait loci (QTL), which made a difference in disease severity of 1.1–1.7 on the standard scoring scale (1–9), were investigated under controlled conditions. The temporal and spatial development of CLS lesions on the two genotypes were monitored using a hyperspectral microscope. The lesion development on the QTL-carrying, resistant genotype was characterised by a fast and abrupt change in spectral reflectance, whereas it was slower and ultimately more severe on the genotype lacking the QTL. An efficient approach for clustering of hyperspectral signatures was adapted in order to reveal resistance characteristics automatically. The presented method allowed a fast and reliable differentiation of CLS dynamics and lesion composition providing a promising tool to improve resistance breeding by objective and precise plant phenotyping.

BackgroundDue to its high damaging potential, Cercospora leaf spot (CLS) caused by Cercospora beticola is a continuous threat to sugar beet production worldwide. Breeding for disease resistance is hampered by the quantitative nature of resistance which may result from differences in penetration, colonization, and sporulation of the pathogen on sugar beet genotypes. In particular, problems in the quantitative assessment of C. beticola sporulation have resulted in the common practice to assess field resistance late in the growth period as quantitative resistance parameter. Recently, hyperspectral sensors have shown potential to assess differences in CLS severity. Hyperspectral microscopy was used for the quantification of C. beticola sporulation on sugar beet leaves in order to characterize the host plant suitability / resistance of genotypes for decision-making in breeding for CLS resistance.ResultsAssays with attached and detached leaves demonstrated that vital plant tissue is essential for the full potential of genotypic mechanisms of disease resistance and susceptibility. Spectral information (400 to 900 nm, 160 wavebands) of CLSs recorded before and after induction of C. beticola sporulation allowed the identification of sporulating leaf spot sub-areas. A supervised classification and quantification of sporulation structures was possible, but the necessity of genotype-specific reference spectra restricts the general applicability of this approach. Fungal sporulation could be quantified independent of the host plant genotype by calculating the area under the difference reflection spectrum from hyperspectral imaging before and with sporulation. The overall relationship between sensor-based and visual quantification of C. beticola sporulation on five genotypes differing in CLS resistance was R2 = 0.81; count-based differences among genotypes could be reproduced spectrally.ConclusionsFor the first time, hyperspectral imaging was successfully tested for the quantification of sporulation as a fungal activity depending on host plant suitability. The potential of this non-invasive and non-destructive approach for the quantification of fungal sporulation in other host–pathogen systems and for the phenotyping of crop traits complex as sporulation resistance is discussed.